Recent research for treatment of ischemic diseases has been performed using growth factors that induce angiogenesis. For example, the therapeutic effect of fibroblast growth factor 2 (FGF2) (Baffour, R. et al., J. Vasc. Surg. 16 (2): 181-91, 1992) and endothelial cell growth factor (ECGF) (Pu, L. Q. et al., J. Surg, Res. 54 (6): 575-83, 1993) on patients with cardiac infarction and acute limb ischemia has been examined. A recent study has revealed that vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) promotes vasculogenesis in animal models with myocardial ischemia and limb ischemia (Takeshita, S. et al., Circulation 90 (5 Pt 2): 11228-34, 1994; Takeshita, S. et al., J. Clin, Invest. 93 (2): 662-70, 1994).
Clinical trials of human gene therapy using angiogenic growth factors have been undertaken recently. Human gene therapy has been clinically applied to therapeutic angiogenesis in order to treat critical ischemic limb. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), an endothelial cell-specific mitogen, is a potent therapeutic gene for this purpose, and it has demonstrated relatively promising results by means of plasmid-based gene transfer involving human subjects (Baumgartner, I., et al., Circulation 97, 1114-1123 (1998); Isner, J. M., et al., J. Vasc. Surg. 28, 964-973 (1998)). However, the related adverse effects and toxicity levels of intramuscular gene transfer of VEGF have been less documented at present because efficiency of plasmid-mediated intramuscular gene transfer and expression are not very high. Since recent reports indicate that transgenic (Thurston, G., et al., Science 286, 2511-2514 (1999)) or adenoviral (Thurston, G., et al., Nature Med. 6, 460-463 (2000)) overexpression of VEGF result in abnormal vasculogenesis in transgene-introduced animals, and that plasmid-based intramuscular VEGF gene transfer showed transient edema in human subjects with ischemic limb (Baumgartner, I., et al., Circulation 97, 1114-1123 (1998); Isner, J. M., et al., J. Vasc. Surg. 28, 964-973 (1998)), detailed mechanisms to cause these pathologies remain to be clarified. Other potential unfavorable effects of VEGF over expression are likely to be the formation of “angioma-like” fragile capillary vessels, possibly due to the imbalance of angiogenic signals (Carmeliet, P., Nature Med. 6, 1102-1103 (2000)). VEGF gene transfer to vessel wall in vivo may cause angiomatousid endothelial proliferation in the severe neointimal formation associating extravasation of red blood cells (Yonemitsu, Y., et al., Lab. Invest. 75, 313-323 (1996)). Similar pathological findings were demonstrated in retrovirus-mediated constitutive overexpression of VEGF in myocardium (Lee, R. J., et al., Circulation 102, 898-901 (2000)). Furthermore, another important issue to be addressed in clinical setting is the level of leakage of locally expressed these angiogenic factors to systemic circulation. Such leakage may cause unexpected angiogenic complications associated with diabetic retinopathy or growth of neoplasm.
Acute critical limb ischemia, which results from acute obstruction of the major arteries, is caused mainly by thrombotic obstruction and is an important target of therapeutic angiogenesis. Acute critical limb ischemia is treated quite unsuccessfully in late interventions, often resulting in limb amputation. Moreover, the long-term prognosis of patients with limb amputation is poor and one-year survival rates of patients after surgery is only 50%. Plasmid-based gene expression levels are low and the efficacy of plasmid-based therapy for acute severe artery occlusion is still unknown.